From past, a method is proposed which introduces an exciting light into a slab-type optical waveguide, propagates the exciting light within the slab-type optical waveguide in a total reflection manner so as to generate an evanescent wave component, excites fluorescent substance by the evanescent wave component which fluorescent substance exist in vicinity of a face of the slab-type optical waveguide which face totally reflects the exciting light, and detects a fluorescent light component by a detector which fluorescent light is a part of the generated fluorescent light and is introduced into the slab-type optical waveguide through the face and is outgone from the slab-type optical waveguide thereafter. When a light propagating within a medium having a larger refractive index totally reflects at a boundary face of the medium and a medium having a smaller refractive index, the evanescent wave is a light component which exist within the medium having smaller refractive index and within a limited region with respect to the boundary face. Specifically, when is is supposed that refractive indexes of the medium are n1, n2 (where n1&lt;n2), a wavelength of the light is .lambda., a incident angle is .theta., an intensity of an incident light is I0, an intensity of a light at a point which is apart from the boundary face by a distance z is I, a specific refractive index is n (=n2/n1), a distance of a point from the boundary face (invasion depth) is dp which point makes an electric field strength to be 1/e of that of the boundary face of the optical waveguide. The intensity I depends upon the distance Z and is expressed with equation (1) . EQU I=I0{4n.sup.2 /(n.sup.2 -1)} cos.sup.2 .theta.exp(-2z/dp) EQU dp=.lambda./2.pi.{(n2.sup.2 /n1.sup.2) sin.sup.2 .theta.-1}.sup.1/2 !(1)
Therefore, it is understood by analyzing the equation (1) that an evanescent wave exists within a limited region with respect to the boundary face (refer tp M. Born & E. Wolf "Principle Of Optics I"). Wherein, it is assumed that the incident angle .theta. is greater than a critical angle .theta.c {.theta.c=sin.sup.-1 (n1/n2)}.
As to evanescent wave, not only penetrating of a light to lower refractive index side but also propagating of a light from lower refractive index side to higher refactive index side satisfy the foregoing. That is, a rate of a light which propagates to fluorescence dye et al. by an angle which is greater than the critical angle is expressed with the equation (1) similarly, which fluorescent dye et al. exists at a position apart from the boundary face by a distance z. Therefore, only light which is generated at vicinity of the boundary face can propagates to the medium having the higher refractive index by an angle which is greater than the critical angle {refer to C. K. Carniglia et. al. "J. Opt. Soc. Am. 62, 479 (1979)}.
It is a phenomenon that evanescent wave exists localized in a region which is near a boundary face and is an extent of nearly wavelength, therefore evanescent wave is possibly applied as a probe light for detecting boundary phenomena. In application to fluoresence immuno assay, determination of reagent labeled with fluorescence dye which dye is bound at a surface of solid phase by antigen-antibody reaction, is possibly performed by directly measuring fluorescent light without separating and washing out unreacted reagent which exist freely within a test liquid.
Following three methods are known as fluorescence immuno assay using above-mentioned evanescent light.
(1) exciting fluorescence dye with evanescent light which dye is bound to a face by immuno-reaction which face is a face of an optical waveguide for totally reflecting an exciting light, and detecting a fluorescent light component by a detector which fluorescent light is a part of fluorescent light radiated from excited fluorescence dye and is intruded into the optical waveguide through the face and is radiated almost uniformly from a face which is opposite to the fluorescent light incidenting face of the optical waveguide as a plane wave thereafter (refer to FIG. 24 and U.S. Pat. No. 3,604,927).
(2) exciting fluorescence dye uniformly using a plane wave, utilizing characteristics in which only fluorescent light from fluorescence dye which are bound to a surface of an optical waveguide, intrudes within the optical waveguide and the fluorescent light propagates within the optical waveguide, and detecting fluorescent light which is outgone from an egde section of the optical waveguide which egde section is located in fluorescent light propagating direction (refer to FIG. 25 and Japanese Patent Laid Open Tokuhyoushou 61-502418), and
(3) exciting fluorescence dye with evanescent light which dye is bound to a face by immuno-reaction which face is a face of an optical waveguide for totally reflecting an exciting light, and detecting a fluorescent light component by a detector which fluorescent light is a part of fluorescent light radiated from fluorescence dye bound to the surface of the optical waveguide and is intruded into the optical waveguide and is propagated within the optical waveguide in a total reflection manner and is outgone from the optical waveguide optical axis of which fluorescent light coincident with that of exciting light (refer to FIG. 26 and Japanese Patent Laid Open Tokuhyoushou 59-501873).